The goal of this proposal is to use zebrafish as a model system to identify novel regulators of ocular lens development and examine how loss of single and combined ?-crystallins, small heat shock proteins important for lens function, affect lens aging and the regulation of lens crystallin expression. These experiments will produce a better understanding of lens development and maintenance of lens transparency important for developing treatments to prevent lens opacities, the leading cause of blindness worldwide. Work in our laboratory has helped make the zebrafish a powerful model system for studying lens development, function and disease. Undergraduate students supported by past funding have cloned and characterized three zebrafish ?-crystallins and used CRISPR/Cas9 gene editing to disable each to examine the impact of their loss on early lens development. Here, we propose in Aim I to use our already produced knockout lines to model the impact of ?-crystallin loss on the maintenance of lens transparency and the gene regulatory networks that control lens protein expression. This aim will include an integration of transcriptomic and proteomic analyses, along with anatomical examination of resulting lens phenotypes. We hypothesize that the resulting lens phenotypes will depend on the specific ?-crystallin lost, providing a novel examination of ?B-crystallin function due to the presence of two orthologs in zebrafish for the single-copy mammalian gene. In Aim II we will use transcriptomic data sets generated by our collaborators from mouse and zebrafish tissues to identify possible novel transcription factors (TFs) regulating lens development. We will then efficiently target these TFs using CRISPR/Cas9 methods that will allow us to screen for lens phenotypes directly in injected embryos. Mutant TF alleles that disrupt normal lens development will be carried into stable lines to detail the role they play in establishing and maintaining lens transparency. We expect that these two aims will efficiently identify novel regulators of lens development and clarify the role of ?- crystallins in maintaining lens transparency. The experiments proposed will provide excellent training opportunities for undergraduates in a research setting with a strong track record of student success.